Transmission of Borrelia burgdorferi (Bb) via tick-bite leads to the development of Lyme disease, which is the leading vector-borne disease in North America. If not diagnosed and treated appropriately with antibiotics, Bb can produce lasting and debilitating disease in myriad tissues, resulting in significant personal and public health costs. After transmission into a host, the bacteria remain locally in the skin tissues for at least 12-48h before disseminating via the skin to eventually establish persistent infection in multiple tissues throughout the host. The mechanisms by which Bb evades immune clearance are largely unknown. In vitro analyses using the different immune cell populations that reside within and immigrate into Bb-infected skin tissues display efficient uptake/killing of Bb, however this spirochete is highly infectious in vivo and readily evades immune clearance, indicating that traditional in vitro analyses are unable to accurately assess critical interactions of these obligae parasites with immune cells in skin. Our group has recently developed novel microscopy techniques that allow for the direct assessment of fluorescent Bb with different immune cell populations directly within the intact skin of living mice and in real-time, providing us for the frst time with the tools to accurately assess these critical interactions in the natural host environment. Preliminary data indicates these techniques can accurately delineate depth and distance in skin tissues, identify different extracellular matrix molecules and their co-localizatin with Bb, and distinguish/measure different diverse motility and interactive characteristics of both Bb and key innate immune cell types, confirming that our techniques are able to perform these analyses. The overall goal is to use these intravital techniques, together with established infection models, to more accurately assess Bb interactions with important immune cell populations and mediators within host skin tissues, and thus more accurately identify virulence mechanisms that are critical for escaping immune clearance and causing Lyme disease. For these studies, fluorescent Bb will be used to describe their dissemination characteristics in skin and use fluorescent knock-in mouse lines to distinguish how several key innate immune cell types respond to Bb infection in skin. The specific aims for this application are: Aim 1. Delineate the importance of Bb motility and chemotaxis properties in evading clearance by immune cells in skin tissues. Aim 2. Delineate whether Bb-elicited IL-10 is responsible for the abbreviated and inefficient innate immune response to Bb infection within skin tissues. Aim 3. Determine the effects of Bb-specific Abs on mediating Bb clearance in skin during early and late stages of infection. These studies should provide the first accurate description of the Bb-immune cell interactions that occur during natural infection and dissemination in skin tissues, and may identify targets for potential Lyme therapies.